AU2018243123B2 - B7-H3 antibody, antigen-binding fragment thereof and medical use thereof - Google Patents
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Abstract
The invention provides a B7-H3 antibody, an antigen-binding fragment thereof and a medical use thereof. Further, the present invention discloses a pharmaceutical composition comprising the B7-H3 antibody or antigen-binding fragment thereof, and the use thereof as a medicament. In particular, the invention discloses a use of a human B7-H3 human antibody or antigen-binding fragment thereof for the manufacture of a medicament for the treatment of a B7-H3-associated disease or condition.
Description
B7-H3 ANTIBODY, ANTIGEN-BINDING FRAGMENT THEREOF AND MEDICAL USE THEREOF
The present invention relates to B7-H3 antibody or antigen-binding fragment thereof, the present invention further relates to a human antibody comprising the CDR region of the B7-H3 antibody, the present invention also relates to a pharmaceutical composition comprising the B7-H3 antibody or antigen-binding fragment thereof, and its use as a diagnostic reagent and therapeutic agent for B7-H3 related diseases.
The T cell-mediated immune response plays an extremely important role in anti-tumor process of the organism, however the activation and proliferation of T cells requires not only the antigen signal recognized by the TCR, but also the second signal provided by co-stimulatory molecules. The molecules of B7 family belong to the co-stimulatory molecule immunoglobulin superfamily. More and more studies have shown that molecules of this family play an important regulatory role in the normal immune function and pathological state in the organism.
B7-H3 is a member of B7 family and belongs to type I transmembrane protein, which contains a signal peptide at the amino terminus, an extracellular immunoglobulin-like variable region (IgV) and constant region (IgC), a transmembrane region, and a cytoplasmic tail region having 45 amino acids (Tissue Antigens. 2007 Aug; 70(2): 96-104). At present, B7-H3 mainly involves two kinds of splicing variants, B7-H3a and B7-H3b. The extracellular domain of B7-H3a consists of two immunoglobulin domains of IgV-IgC (also known as 2IgB7-H3), while the extracellular domain of B7-H3b consists of four immunoglobulin domains of IgV-IgC-IgV-IgC (also known as 4IgB7-H3).
B7-H3 protein is not expressed or poorly expressed in normal tissues and cells, but highly expressed in various tumor tissues and is closely related to tumor progression, patient survival and prognosis. It has been clinically reported that B7-H3 is over-expressed in many types of cancers, especially in non-small cell lung cancer, renal cancer, urinary tract epithelial cancer, colorectal cancer, prostate cancer, glioblastoma multiforme, ovarian cancer and pancreas cancer (Lung Cancer. 2009 Nov; 66(2): 245-249; Clin Cancer Res. 2008 Aug 15; 14(16): 5150-5157). In addition, it has also been reported in the literature that, in prostate cancer, the expression level of B7-H3 is positively correlated with clinical pathological malignancy (such as tumor volume, extra-prostatic invasion or Gleason score), and is also associated with cancer progression (Cancer Res. 2007 Aug 15; 67(16):7893-7900). Similarly, in glioblastoma multiforme, the expression of B7-H3 is inversely associated with event-free survival, and in pancreatic cancer, the expression of B7-H3 is associated with lymph node metastasis and pathological progression. Therefore, B7-H3 is considered as a new tumor marker and potential therapeutic target.
Currently, there have been therapeutic strategies specific for B7-H3 target for preclinical studies, for example, antibodies targeting murine B7-H3 will enhance infiltrative CD8-positive T cells in tumor and inhibit tumor growth (Mod Pathol. 2010 Aug; 23(8): 1104-1112). Furthermore, patent WO 2008/066691 shows that antibodies recognizing the B7-H3 variant B7-H3a exhibited an in vivo anti-tumor effect on adenocarcinoma. In clinical studies, an ADC of murine B7-H3 antibody conjugated with radioactive 131 significantly inhibited the growth of neuroblastoma in patients (J Neufooocol 97(3):409-18 (2010)). However, the B7H3 antibodies currently under study are humanized antibodies that have been engineered by humanization of murine antibodies. However, humanized antibodies upon immunization have higher immunogenicity than fully human antibodies which do not contain any murine antibody components. It is an unfavorable factor in human application.
Phage display technology refers to the fusion of an exogenous protein or polypeptide with a phage coat protein, so as to express an exogenous protein on the surface of the phage. The phage antibody library is an antibody library established by combining phage display technology, PCR amplification technology and protein expression technology.
The biggest advantage of the phage antibody library is to prepare the fully human antibody by mimicking the three processes of antibody production in vivo without immunization in vivo. In addition, the phage antibody library has the following advantages: 1) The unification of genotype and phenotype is achieved; in addition, the experimental method is simple and rapid, whereas the traditional antibody production method by hybridoma technology takes several months; but in contrast the antibody library technology takes only a few weeks. 2) The expressed product is a fully human antibody, and the molecular weight thereof is small, the antibody is mainly expressed in the form of active fragments Fab and scFv; when compared with complete antibody, it has obvious advantages in tissue penetrability. 3) Screening capacity is large: hybridoma technology is used to screen among thousands of clones, but antibody library technology can be used to select from millions or even hundreds of millions of molecules; therefore more types of antibodies will be obtained. 4) Wide application: utilizing prokaryotic expression system leads to more obvious advantage in large scale production (Curr Opin Biotechnol. 2002 Dec;13(6):598-602; Immunotechnology, 2013, 48(13) 48(13): 63-73).
At present, patents such as W02008100934, W02010096734, W02012147713, W02015181267, W02016044383 etc. have reported B7-H3 antibodies, however, most of them are murine antibodies or humanized antibodies; and most of them are still in clinical phase I and discovery phase, either in domestic or overseas, none of antibody drug targeting B7-H3 is available in market; so it is necessary to further develop B7-H3 fully human antibody with higher activity, high affinity and high stability, for the treatment of related diseases and application.
One purpose of the present invention is to provide a monoclonal antibody or antigen-binding fragment thereof that binds to the amino acid sequence(s) or three-dimensional structure of the B7-H3 extracellular region. Furthermore, another purpose of the present invention is to screen and obtain highly active and highly stable anti-human B7-H3 fully human antibodies that compete with the monoclonal antibody or antibody fragments thereof.
Furthermore, the present invention provides a DNA encoding the antibody, a vector comprising the DNA, a transformant obtained by transforming the vector, a method of producing an antibody or antibody fragment thereof using the transformant, and a diagnostic reagent or therapeutic agent in which said antibody or antibody fragment thereof is served as active ingredient.
In one aspect, the present invention provides a B7-H3 antibody or antigen-binding fragment thereof which binds to human B7-H3, wherein the B7-H3 antibody or antigen-binding fragment thereof is selected from any one of the monoclonal antibody or antigen-binding fragment thereof of the following (i) to (ii):
(i) a monoclonal antibody or antigen-binding fragment thereof, comprising one or more CDR region sequences selected from the following sequences or selected from the amino acid sequences with at least 95% identity to the following:
antibody heavy chain variable region HCDR sequences as shown in SEQ ID NO: 10, 11 and 12; and antibody light chain variable region LCDR amino acid sequences as shown in SEQ ID NO: 13, 14 and 15;
(ii) a monoclonal antibody or antigen-binding fragment thereof, comprising one or more CDR region sequences selected from the following sequences or selected from the amino acid sequences with at least 95% identity to the following: antibody heavy chain variable region HCDR sequences as shown in SEQ ID NO: 16, 17 and 18; and antibody light chain variable region LCDR sequences as shown in SEQ ID NO: 19, 20 and 21.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the monoclonal antibody is a recombinant antibody.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the monoclonal antibody is a human recombinant antibody or antigen-binding fragment thereof.
In a preferred embodiment, the B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the framework (FR) sequences of light chain and the heavy chain variable regions of human recombinant antibody are derived from a human germline light chain and heavy chain, respectively, or mutant sequence thereof.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the human recombinant antibody comprises a heavy chain variable region as shown in SEQ ID NO: 6 or 8 or variant thereof; wherein the variant has 1-10 amino acid substitution(s) in the heavy chain variable region sequence as shown in SEQ ID NO: 6 or 8.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the human recombinant antibody comprises a light chain variable region as shown in SEQ ID NO: 7 or 9 or variant thereof; wherein the variant has 1-10 amino acid substitution(s) in the light chain variable region sequence of SEQ ID NO: 7 or 9.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the B7-H3 antibody further comprises a human antibody constant region, preferably the B7-H3 antibody is a full-length antibody consisting of the heavy chain and light chain sequence as shown in SEQ ID NO: 22 and 23, respectively; or a full-length antibody consisting of the heavy chain and light chain sequence as shown in SEQ ID NO: 22 and 26, respectively; or a full-length antibody consisting of the heavy chain and light chain sequence as shown in SEQ ID NO: 24 and 25, respectively.
In a preferred embodiment, a B7-H3 antibody or antigen-binding fragment thereof according to the present invention is provided, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv) and CDR-containing peptide.
In another aspect, the present invention provides an isolated B7-H3 antibody or antigen-binding fragment thereof, characterized in that it competes with the B7-H3 antibody or antigen-binding fragment thereof as described above for binding to human B7-H3.
The present invention also provides a pharmaceutical composition comprising a therapeutically effective amount of the B7-H3 antibody or antigen-binding fragment thereof according to the present invention, and one or more pharmaceutically acceptable carrier, diluent or excipient.
The present invention also provides a nucleic acid molecule encoding the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention.
The present invention also provides a recombinant vector comprising the nucleic acid molecule as described above.
The present invention also provides a host cell transformed with the recombinant vector as described above, the host cell is selected from the group consisting of prokaryotic cell and eukaryotic cell, preferably eukaryotic cell, more preferably mammalian cell or yeast cell.
The present invention also provides a method for producing the antibody or antigen-binding fragment thereof according to present invention, wherein the method includes culturing the host cell as described above in a culture to form and accumulate the B7-H3 antibody or antigen-binding fragment thereof according to present invention, and recovering the accumulated antibody or antigen-binding fragment thereof from the culture.
The present invention also provides a method for immunologically detecting or measurement of B7-H3, wherein the method comprises utilizing the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention.
The present invention also provides a reagent for detecting or measurement of human B7-H3, wherein the reagent comprises the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention.
The present invention also provides a diagnostic reagent for detecting diseases associated with B7-H3 positive cells, the diagnostic reagent comprises the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention.
The present invention also provides a method for diagnosing diseases related to B7-H3 positive cells, the method comprises detecting or determining B7-H3 or B7-H3 positive cells by using the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention.
In another aspect, the present invention also provides the use of the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention for the preparation of a diagnostic reagent for diseases related to B7-H3 positive cells.
In another aspect, the present invention further provides a therapeutic agent for treating diseases associated with B7-H3 positive cells, the therapeutic agent comprises the B7-H3 antibody or antigen-binding fragment thereof as described in the present invention, or comprises the pharmaceutical composition as described above, or the nucleic acid molecule as described above.
The present invention also provides a method for treating diseases related to B7-H3 positive cells, the method comprises inducing cell death of B7-H3 positive cells by utilizing the antibody or antigen-binding fragment thereof as described in the present invention, or the pharmaceutical composition as described above, or the nucleic acid molecule as described above.
In another aspect, the present invention further provides the use of the antibody or antigen-binding fragment thereof, or the pharmaceutical composition, or the nucleic acid molecule as described in the present invention in the preparation of therapeutic agents for treating diseases related to B7-H3 positive cells.
Figure 1: Binding ability of different antibodies to human 2Ig-B7-H3 antigen;
Figure 2: Binding ability of different antibodies to human 4Ig-B7-H3 antigen;
Figure 3: Cross-binding ability of different antibodies to murine B7-H3 antigen;
Figure 4: Binding ability of different antibodies to U87MG cells;
Figure 5: Endocytic effect of different antibodies on U87MG cells.
1. TERMS
In order to more readily understand the present invention, certain technical and scientific terms are specifically defined below. Unless specifically indicated elsewhere in this document, all other technical and scientific terms used herein have the meaning commonly understood by one of ordinary skilled in the art to which this invention pertains.
As used herein, the three-letter code and the single-letter code for amino acids are as described in J. biol. chem, 243, p 3 5 5 8 (1968).
As used herein, "antibody" refers to immunoglobulin, a structure of four-peptide chains connected together by disulfide bonds between two identical heavy chains and two identical light chains. Different immunoglobulin heavy chain constant regions exhibit different amino acid compositions and rank orders, hence present different kinds of antigenicity. Accordingly, immunoglobulin can be divided into five categories, or called as immunoglobulin isotypes, namely IgM, IgD, IgG, IgA and IgE, with heavy chain p, 6, y, aand s, respectively. According to its amino acid composition of hinge region and the number and location of heavy chain disulfide bonds, the same type of Ig can be divided into different sub-categories, for example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4. Light chains can be divided into K or k chain, due to different constant regions. Each IgG among the five types has K or k chain.
In the present invention, the antibody light chain as described herein further comprises a light chain constant region, wherein the light constant region comprises a human or murine K, k chain or a variant thereof.
In the present invention, the antibody heavy chain as described herein further comprises a heavy chain constant region, wherein the heavy chain constant region comprises human or murine IgGI, IgG2, IgG3, IgG4 or a variant thereof.
The sequence of about 110 amino acids close to the N-terminus of the antibody heavy and light chains, changes largely, known as variable region (Fv region); the rest sequence of amino acids close to the C-terminus is relatively stable, known as constant region. Variable region comprises three hypervariable regions (HVR) and four framework regions (FR) having relatively conserved sequence. Three hypervariable regions determine the specificity of the antibody, also known as complementarity determining region (CDR). Each light chain variable region (LCVR) and each heavy chain variable region (HCVR) is composed of three CDR regions and four FR regions, sequentially order from the amino terminus to the carboxyl terminus is: FRI, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Three light chain CDRs refer to LCDR1, LCDR2, and LCDR3; three heavy chain CDRs refer to HCDR1, HCDR2 and HCDR3. The number and location of CDR amino acid residues in LCVR and HCVR regions of the antibody or antigen binding fragment herein comply with known Kabat numbering criteria (LCDR1-3, HCDR2-3), or comply with Kabat and Chothia numbering criteria (HCDR1).
The terms "human antibody" and "human derived antibody" are used interchangeably and refer to an antibody comprises one or more variable and constant regions are derived from human immunoglobulin sequence. One of the preferred ways is that all of the variable and constant regions are derived from human immunoglobulin sequences, i.e., "fully human derived antibody" or "fully human antibody". These antibodies can be obtained in a variety of ways, including antibodies obtained by using phage display technology, includes isolating B cells from human PBMC, spleen, lymph node tissue and construct natural single-stranded phage human antibody library, or by immunizing transgenic mice expressing human antibody light and heavy chain and screening.
The term "murine antibody" used in the present invention refers to a monoclonal antibody against human B7-H3 prepared according to the knowledge and skill in the art. During preparation, the test subject is injected with B7-H3 antigen, and then the hybridoma expressing antibodies having desired sequence or functional properties are isolated. In a preferred embodiment of the invention, the murine B7-H3 antibody or antigen-binding fragment thereof further comprises a light chain constant region of a murine kappa, lambda chain or a variant thereof, or further comprises a heavy chain constant region of murine IgG1, IgG2, IgG3 or a variants thereof.
The term "chimeric antibody", is an antibody which is formed by fusing the variable region of a murine antibody with the constant region of human antibody, so as to alleviate the murine antibody-induced immune response. To establish a chimeric antibody, a hybridoma secreting a specific murine monoclonal antibody is established, and a variable region gene is cloned from murine hybridoma cells, and then a desired constant region gene of human antibody is cloned, and connected with the murine variable region gens to form a chimeric gene which can be subsequently inserted into expression vector, and finally the chimeric antibody molecule is expressed in eukaryotic or prokaryotic system. In a preferred embodiment of the present invention, the light chain of the B7-H3 chimeric antibody further comprises light chain constant region derived from human kappa, lambda chain or variant thereof. The heavy chain of the B7-H3 chimeric antibody further comprises heavy chain constant region derived from human IgGI, IgG2, IgG3 or IgG4 or a variant thereof, and preferably comprises heavy chain constant region derived from human IgG, IgG2 or IgG4, or variant of IgGI, IgG2 or IgG4 with amino acid mutation (such as YTE mutation or back mutation).
The term "humanized antibody", also known as CDR-grafted antibody, refers to an antibody generated by grafting murine CDR sequences into a variable region framework of a human antibody (i.e. antibodies produced within different types of human germline antibody framework sequences). Humanized antibody overcomes the heterologous response induced by the chimeric antibody that carries a large amount of murine protein components. Such framework sequences can be obtained from public DNA databases including germline antibody gene sequences or published references. For example germline DNA sequences of human heavy and light chain variable region genes can be found in e.g. VBase" human germline sequence database (available on the Internet at www.mrccpe.com.ac.uk/vbase), as well as found in Kabat, EA, et al, 1991 Sequences of Proteins of Immunological Interest, 5th edition.
The CDR graft can reduce the affinity of the B7-H3 antibody or antigen-binding fragment thereof to the antigen, due to the framework residues that are in contact with the antigen. Such interaction can be the result of hyper-mutation in somatic cells. Therefore, it may still be necessary to graft such donor framework amino acids onto the framework of humanized antibodies. Amino acid residues from non-human B7-H3 antibody or antigen-binding fragment thereof which are involved in antigen binding can be identified by examining the murine monoclonal antibody variable region sequences and structures. Each residue in the CDR donor framework that differs from the germline can be considered to be relevant. If the closest germline cannot be determined, the sequence can be compared with the common sequence of a subtype or the sequence of the murine with high similarity percentage. Rare framework residues are thought to be the result of somatic hyper-mutation and thus play an important role in binding.
The term "antigen-binding fragment" or "functional fragment" of an antibody refers to one or more fragments of antibody that retain the ability to specifically bind to an antigen (eg, B7-H3). It has been shown that fragments of full-length antibodies can be used for antigen binding function of antibodies. Examples of the binding fragments included in the term "antigen-binding fragment" of an antibody include: (i) Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHI domain; (ii) F(ab') 2 fragment, a bivalent fragment comprising two Fab fragments linked by disulfide bonds in the hinge region, (iii) Fd fragment, consisting of the VH and CHI domain; (iv) Fv fragment, consisting of the VH and VL domain of one arm of the antibody; (v) single domain or dAb fragment (Ward et al. (1989) Nature 341: 544-546) composed of VH domain; and (vi) a separate complementarity determining region (CDR); or (vii) a combination of two or more separated CDRs optionally linked by a synthetic linker. Furthermore, although VL domain and VH domain of Fv fragment are encoded by separated genes, they can be linked by synthetic linker using recombinant method such that they can generating a single protein chain with monovalent molecular by pairing the VL and VH domain (called single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci USA 85: 5879-5883). Such single chain antibodies are also intended to be included in the term "antigen-binding fragment" of an antibody. Such antibody fragments are obtained using conventional techniques known in the art, and functional screening of fragments are used in the same way as the intact antibodies. The antigen binding sites can be produced by recombinant DNA techniques or by enzymatic or chemical disruption of the intact immunoglobulin. The antibodies may be in different phenotype, e.g., IgG (e.g., IgG1, IgG2, IgG3 or IgG4 subtype), IgAl, IgA2, IgD, IgE or IgM antibody.
The antigen-binding fragments of the present invention include Fab, F(ab') 2 , Fab', single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv), CDR-containing peptide, etc.
Fab is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity, such fragment is obtained by treating an IgG antibody molecule with protease papain (cleaving amino acid residue at position 224 of H chain), wherein about half of the N-terminal side of H chain and the entire L chain are bound by disulfide bond.
The Fab of the present invention can be produced by treating the monoclonal antibodies of the present invention which specifically recognize human B7-H3 and bind to the extracellular region amino acid sequence or three-dimensional structure thereof with papain. Furthermore, the Fab can be produced by inserting a DNA encoding Fab of the antibody into a prokaryotic expression vector or eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab.
F(ab') 2 is an antibody fragment obtained by digesting the lower part of two disulfide bonds in IgG hinge region with pepsin. It has a molecular weight of about 100,000 and antigen-binding activity, and comprises two Fab regions linked at the hinge position.
The F(ab')2 of the present invention can be produced by treating the monoclonal antibody of the present invention which specifically recognize human B7-H3 and bind to the extracellular region amino acid sequence or three-dimensional structure thereof with pepsin. Furthermore, the F(ab') 2 can be produced by linking the Fab' described below with thioether bond or disulfide bond.
Fab' is an antibody fragment having a molecular weight of about 50,000 and having antigen-binding activity. It is obtained by cleaving the disulfide bond in the hinge region of the F(ab') 2 mentioned above. The Fab' of the present invention may be produced by treating the F(ab')2 of the present invention which specifically recognize human B7-H3 and bind to the extracellular region amino acid sequence or three-dimensional structure thereof with reducing agent (such as dithiothreitol).
Furthermore, the Fab' can be produced by inserting a DNA encoding Fab' fragment of antibody into prokaryotic expression vector or eukaryotic expression vector and introducing the vector into prokaryote or eukaryote to express the Fab'.
The term "single-chain antibody", "single-chain Fv" or "scFv" refers to a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules have the general structure: NH2-VL-linker-VH-COOH or NH2-VH-linker-VL-COOH. Suitable linkers in prior art consist of repeated GGGGS amino acid sequence or variants thereof, for example variant having 1-4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90: 6444 -6448). Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8: 725-731, Choi et al. (2001), Eur. J. Immunol. 31: 94-106, Hu et al. (1996), Cancer Res. 56: 3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293: 41-56 and Roovers et al. (2001), Cancer Immunol.
The scFv of the present invention can be produced by following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7-H3 and binds to extracellular region amino acid sequence or three-dimensional structure thereof; constructing a DNA encoding the scFv; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said scFv.
A diabody is an antibody fragment in which the scFv is dimerized, and is an antibody fragment having bivalent antigen-binding activity. In the bivalent antigen binding activity, the two antigens may be the same or different.
The diabody of the present invention can be produced by following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7-H3 and binds to extracellular region amino acid sequence or three-dimensional structure thereof; constructing DNA encoding scFv such that the length of the linker peptide is 8 or less amino acid residues; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express the diabody.
The dsFv is obtained by substituting one amino acid residue in each of VH and VL with cysteine residue, and then linking the polypeptides via disulfide bond between the two cysteine residues. The amino acid residue to be substituted with a cysteine residue can be selected based on antibody three-dimensional structure prediction of the antibody in accordance with known method (Protein Engineering, 7, 697 (1994)).
The dsFv of the present invention can be produced by following steps: obtaining the cDNA encoding VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7-H3 and binds to extracellular region amino acid sequence or three-dimensional structure thereof; constructing dsFv-encoding DNA; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said dsFv.
CDR-containing peptide is constructed by one or more regions of CDR of VH or VL. Peptides comprising several CDRs can be linked directly or via suitable peptide linker.
The CDR-containing peptide of the present invention can be produced by following steps: constructing DNA encoding CDRs of VH and VL of the monoclonal antibody of the present invention which specifically recognizes human B7-H3 and binds to extracellular region amino acid sequence or three-dimensional structure thereof; inserting the DNA into prokaryotic expression vector or eukaryotic expression vector; and then introducing the expression vector into prokaryote or eukaryote to express said peptide. The CDR-containing peptide can also be produced by chemical synthesis methods such as Fmoc method or tBoc method.
The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily contributes to antigen binding. One of the most commonly used definitions for the six CDRs is provided by Kabat E.A. et al. (1991) Sequences of proteins of immunological interest. NIH Publication 91-3242. As used herein, the Kabat definition of CDR only applies to CDR1, CDR2 and CDR3 of light chain variable domain (CDR L, CDR L2, CDR L3 or LI, L2, L3), as well as CDR2 and CDR3 of heavy chain variable domain (CDR H2, CDR H3 or H2, H3).
The term "antibody framework" as used herein, refers to a portion of the variable domain VL or VH, which serves as a scaffold for the antigen binding loop (CDR) of the variable domain. Essentially, it is a variable domain without CDRs.
The term "epitope" or "antigenic determinant" refers to a site on an antigen to which an immunoglobulin or antibody specifically binds (e.g., a specific site on B7-H3 molecule). Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).
The terms "specific binding", "selective binding", "selectively bind" and "specifically bind" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10-7 M, such as approximately less than about 10-8 M, 10-9 M or 10-10M or less.
The term "KD" or "Kd" refers to the dissociation equilibrium constant for particular antibody-antigen interaction. Typically, the antibody of the present invention binds to B7-H3 with a dissociation equilibrium constant (KD) of less than about 10-7 M, such as less than about 10-8 M, 10-9 M or 10-10 M or less, for example, as determined using surface plasmon resonance (SPR) techniques in a BIACORE instrument.
The term "competitive binding" refers to that an antibody recognizes and binds to the same epitope (also known as antigenic determinant) or a portion thereof of the extracellular domain of human B7H3 as the one recognized by monoclonal antibody of the present invention. An antibody that binds to the same epitope as the monoclonal antibody of the present invention refers to an antibody that recognizes and binds to the amino acid sequence of human B7-H3 recognized by the monoclonal antibody of the present invention.
The term "nucleic acid molecule" as used herein refers to DNA molecule and RNA molecule. The nucleic acid molecule may be single stranded or double stranded, but is preferably a double stranded DNA. A nucleic acid is "effectively linked" when it is placed into functional relationship with another nucleic acid sequence. For example, if a promoter or enhancer affects transcription of a coding sequence, the promoter or enhancer is effectively linked to the coding sequence.
The term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. In one embodiment, the vector is a "plasmid" which refers to a circular double stranded DNA loop into which additional DNA segment can be ligated. In another embodiment, the vector is a viral vector, wherein additional DNA segment can be ligated into viral genome. The vectors disclosed herein are capable of self-replicating in host cell into which they have been introduced (for example, a bacterial vector having bacterial replication origin and episomal mammalian vector) or can be integrated into the genome of host cell upon introduction into host cell, thereby is replicated along with the host genome (e.g., a non-episomal mammalian vector).
Methods for producing and purifying antibodies and antigen-binding fragments are well known in the art, such as Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15. For example, mice can be immunized with human B7-H3 or a fragment thereof, and the obtained antibody can be re-natured, purified, and sequenced by using conventional method known in the art. The antigen-binding fragment can also be prepared by conventional method. The antibodies or antigen-binding fragments of the invention are genetically engineered to add one or more human FR regions in non-human CDR region. The human FR germline sequence(s) can be obtained by aligning human antibody variable germline gene database and MOE software from the ImMunoGeneTics (IMGT) website at http://imgt.cines.fr or from the Journal of Immunoglobulins 2001ISBN012441351.
The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells can include bacterial, microbial, plant or animal cells. Bacteria susceptible to be transformed include members of the Enterobacteriaceaefamily, such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; Pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO (Chinese hamster ovary cell line) and NSO cells.
The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. For example, cDNA sequence(s) encoding a heavy chain and a light chain can be cloned and recombined into GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected with CHO cells. As a more recommended prior art, mammalian expression systems result in glycosylation of antibodies, particularly at the highly conserved N-terminal site in the Fc region. Stable clones are obtained by expressing antibodies that specifically bind to human B7-H3. Positive clones are expanded in serum-free medium in a bioreactor to produce antibodies. The culture medium containing secreted antibody can be purified by conventional technique. For example, purification is carried out using A or G Sepharose FF column that has been equilibrated with a compatible buffer. The non-specifically bound components are removed by washing. The bound antibody is eluted by a pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated by a conventional manner. Soluble aggregate and multimers can also be removed by conventional methods such as size exclusion or ion exchange. The product needs to be frozen immediately, such as at -70 °C, orlyophilized.
"Administration" and "treatment", when applied to an animal, human, experimental subject, cell, tissue, organ, or biological fluid, refer to contact an exogenous pharmaceutical, therapeutic, diagnostic reagent, or composition with the animal, human, subject, cell, tissue, organ, or biological fluid. "Administration" and "treatment" can refer, e.g., to therapeutic, pharmacokinetic, diagnostic, research, and experimental methods. Treatment of a cell encompasses contacting a reagent with the cell, as well as contacting a reagent with a fluid, wherein the fluid is in contact with the cell. "Administration" and "treatment" also mean in vitro and ex vivo treatments, e.g., of a cell, by a reagent, diagnostic, binding composition or by another cell. "Treatment", when applied to a human, veterinary, or research subject, refers to therapeutic treatment, prophylactic or preventative measures, research and diagnostic applications.
"Treat" means to administer a therapeutic agent, such as a composition comprising any of the binding compounds of the present invention, internally or externally to a patient having one or more disease symptoms for which the agent has known therapeutic activity. Typically, the agent is administered in an amount effective to alleviate one or more disease symptoms in the treated patient or population, so as to induce the regression of such symptom(s) or to prevent the progression by any clinically measurable degree. The amount of a therapeutic agent that is effective to alleviate any particular disease symptom (also referred to "therapeutically effective amount") may vary according to factors such as the disease state, age and weight of the patient, and the ability of the agent to elicit desired response in the patient. Whether a disease symptom has been alleviated can be assessed by any clinical measurement typically used by physicians or other skilled healthcare providers to assess the severity or progression status of that symptom. Even if an embodiment of the present invention (e.g., a treatment method or article of manufacture) is not be effective in alleviating the target disease symptom(s) in every patient, it should alleviate the target disease symptom(s) in a statistically significant number of patients as determined by any statistical test known in the art such as the Student's t-test, chi-square test, U-test according to Mann and Whitney, Kruskal-Wallis test (H-test), Jonckheere-Terpstra test and Wilcoxon test.
"Conservative modification" or "conservative replacement or substitution" refers to substitutions of amino acids in a protein with other amino acid having similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation and rigidity, etc.), such that the changes can be frequently made without altering the biological activity of the protein. It will be appreciated by those skilled in the art that, in general, a single amino acid substitution in non-essential region of polypeptide does not substantially alter biological activity (see, for example, Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th edition)). In addition, substitutions with structurally or functionally similar amino acids are unlikely to disrupt biological activity.
An "effective amount" includes an amount sufficient to ameliorate or prevent a symptom or condition of medical disease. An effective amount also means an amount sufficient to allow or facilitate the diagnosis. An effective amount for a particular patient or veterinary subject can vary depending on factors such as: the condition to be treated, the overall health condition of the patient, the route and does of administration, and the severity of side effects. An effective amount can be the maximum dose or dosing regimen that avoids significant side effects or toxic effects.
"Exogenous" refers to a substance that is produced outside of organism, cell or human, depending on the situation. "Endogenous" refers to a substance that is produced in a cell, organism or human, depending on the situation.
"Identity" refers to the sequence similarity between two polynucleotide sequences or two polypeptide sequences. When the positions in two sequences to be compared are occupied by the same base or amino acid monomer subunit, for example, if each position of two DNA molecules is both occupied by adenine, the molecules are considered to be homologous at that position. The percent identity between the two sequences is a function of the number of matched or homologous positions shared by two sequences divided by the number of positions to be compared x 100. For example, in the optimal alignment of sequence(s), if there are 6 matches or homologs among 10 positions between two sequences, then the two sequences are deemed as 60% homology; if there are 95 matches or homologs among 100 positions between two sequences, then the two sequences are deemed as 95% homology. In general, a comparison is performed when the maximum identity percentage is obtained by aligning two sequences.
As used herein, the expressions "cell", "cell line" and "cell culture" are used interchangeably, and all such terms include the progeny thereof. Thus, the words "transformant" and "transformed cell" include primary test cells and cultures derived therefrom, regardless of the number of passages. It should also be understood that all progeny may not be exactly identical in terms of DNA content due to deliberate or inadvertent mutations. The mutant progeny having the same function or biological activity as screened for the primarily transformed cell is included. In the case of a different name, it is clearly understood from the context.
As used herein, "polymerase chain reaction" or "PCR" refers to a procedure or technique in which small amount of particular portion of nucleic acid, RNA, and/or DNA are amplified as described for example in U.S. Patent No. 4,683,195. In general, it is necessary to obtain sequence information from the end or beyond the target region, thereby oligonucleotide primers can be designed; these primers are identical or similar to the opposite strands of the template to be amplified. The 5' terminal nucleotides of the two primers may be identical to the ends of the material to be amplified. PCR can be used to amplify specific RNA sequences, specific DNA sequences from total genomic DNA, and cDNA transcribed from total cellular RNA, phage or plasmid sequences, etc. See generally, Mullis et al. (1987) Cold Spring Harbor Symp. Ouant. Biol. 51:263;
Erlich ed., (1989) PCR TECHNOLOGY (Stockton Press, N.Y.). The PCR used herein is considered as an example (but not the only one) of nucleic acid polymerase reaction method for amplifying a nucleic acid test sample, said method comprises the use of known nucleic acid as a primer and nucleic acid polymerase to amplify or produce specific portion of the nucleic acid.
"Optional" or "optionally" means that the event or situation described subsequently may (but need not to) occur, this includes where the event or situation occurs or does not occur. For example, "optionally comprising 1-3 antibody heavy chain variable regions" means that the antibody heavy chain variable region with specific sequence can be, but need not to, be present.
"Pharmaceutical composition" means a mixture comprising one or more compounds described herein or physiologically/pharmaceutically acceptable salt or prodrug thereof, along with other chemical components, such as physiological/pharmaceutically acceptable carrier and excipient. The purpose of the pharmaceutical composition is to promote the administration to the organism, and facilitate the absorption of active ingredient and thereby exerting a biological activity.
Furthermore, the present invention relates to a method for immunologically detecting or determining B7-H3, a reagent for immunologically detecting or determining B7-H3, a method for immunologically detecting or determining cells expressing B7-H3, and a diagnostic reagent for diagnosis of disease related to B7-H3 positive cells, comprising the monoclonal antibody or antibody fragment of the present invention that specifically recognizes human B7-H3 and binds to extracellular region amino acid sequence or three-dimensional structure thereof, as an active ingredient.
In the present invention, the method for detecting or determining the amount of B7-H3 may be any known method. For example, it includes immunodetection or assay.
The immunodetection or assay is a method of detecting or determining the amount of antibody or antigen by using labeled antigen or antibody. Examples of immunodetection or assay include radioactive substance labeled immunological antibody method (RIA), enzyme immunoassay (EIA or ELISA), fluorescent immunoassay (FIA), luminescent immunoassay, western blotting method, physicochemical methods, etc.
The above-mentioned diseases related to B7-H3 positive cells can be diagnosed by detecting or determining cells expressing B7-H3 by using the monoclonal antibodies or antibody fragments thereof of the present invention.
In order to detect cells expressing the polypeptide, a known immunodetection can be used, and preferably immunoprecipitation, fluorescent cell staining or immunohistochemical staining etc. can be used. Furthermore, a fluorescent antibody staining method etc. using FMAT8100HTS system (Applied Biosystem) can be used.
In the present invention, a living sample for detecting or determining B7-H3 is not particularly limited, so as long as it has a possibility of including cells expressing B7-H3, such as tissue cells, blood, plasma, serum, pancreatic fluid, urine, feces, tissue fluid or culture fluid.
The diagnostic reagent containing the monoclonal antibody or the antibody fragment thereof of the present invention may further contain a reagent for performing antigen-antibody reaction or a reagent for detecting the reaction, depending on desired diagnostic method. The reagent for performing antigen-antibody reaction includes buffers, salts, and the like. The reagent for detection includes reagents commonly used in immunodetection or measurement, such as labeled secondary antibodies that recognize the monoclonal antibodies, antibody fragments or conjugates thereof, substrates corresponding to the labels, etc.
The present invention is also relates to method of treating diseases relates to human B7-H3 positive cells, particularly in the treatment of cancer and inflammation.
2. EXAMPLES AND TEST EXAMPLES
The present invention is further described below in conjunction with the example, however the scope of the present invention is not limited thereto. .In the examples of the present invention, where specific conditions are not described, the experimental are generally conducted under conventional conditions as described in Cold Spring Harbor Antibody Technology Laboratory Manual, Molecular Cloning Manual, or under conditions recommended by the manufacturer of raw material or products. Where the source of the reagents is not specifically given, the reagents are commercially available conventional reagents.
Example 1. Preparation of B7-H3 antigen and protein for detection
Design of B7-H3 antigen
The human B7-H3 as shown in SEQ ID NO: 1 was used as the template for B7-H3 of the present invention, and the amino acid sequence of the antigen and protein for detection involved in the present invention were designed. Unless otherwise specified, the following B7-H3 antigen is human B7-H3.
Human B7-H3 full-length protein: B7-H3 (SEQ ID NO: 1):
Note:
The double-underlined portion is the signal peptide (Signal peptide: 1-28);
The underlined portion is the B7-H3 extracellular domain (Extracellular domain: 29-466), wherein 29-139 is Ig-like V-type 1 Domain, and 145-238 is Ig-like C2-type 1 Domain; 243-357 is Ig-like V-type 2 Domain, 363-456 is Ig-like C2-type 2 Domain;
The dot-lined portion is the transmembrane domain portion (Transmembrane domain: 467-487);
The italic portion is the intracellular domain (Cytoplasmic domain: 488-534).
Murine B7-H3 full-length amino acid sequence (SEQ ID NO: 2)
MLRGWGGPSVGVCVRTALGVLCLCLTGAVEVOVSEDPVVALVDTDATLRCSF SPEPGFSLAOLNLIWOLTDTKOLVHSFTEGRDQGSAYSNRTALFPDLLVQGNAS LRLORVRVTDEGSYTCFVSIQDFDSAAVSLOVAAPYSKPSMTLEPNKDLRPGN MVTITCSSYOGYPEAEVFWKDGOGVPLTGNVTTSOMANERGLFDVHSVLRVV LGANGTYSCLVRNPVLQODAHGSVTITGOPLTFPPEALWVTVGLSVCLVVLLV .AAFVCWRKIKQSCEEENAGAEDQDGDGEGSKTALRPLKPSENKEDDGQEiA
Note:
The double-underlined portion is the signal peptide (Signal peptide: 1-28);
The underlined portion is the B7-H3 extracellular domain (Extracellular domain: 29-248), wherein 29-139 is Ig-like V-type Domain, and 145-238 is Ig-like C2-type Domain;
The dot-lined portion is the transmembrane domain portion (Transmembrane domain:
249-269);
The italic portion is the intracellular domain (Cytoplasmic domain: 270-316).
The human B7-H3 antigen (SEQ ID NO: 3) used for screening and detection is a commercial product (R&D cat# 1949-B3-050/CF, abbreviated as 2g-B7-H3), and sequence is as follows:
Note: The underlined portion is the B7-H3 extracellular region; the italic portion is the His-tag marker.
The human B7-H3 antigen (SEQ ID NO: 4) used for detection is a commercial product (SinoBiological cat # 11188-H08H, abbreviated as 41g-B7-H3), and the sequence is as follows:
Note: The underlined portion is the B7-H3 extracellular region; the italic portion is the His-tag marker.
The murine B7-H3 antigen (SEQ ID NO: 5) used for screening and detection is a commercial product (R&D cat#1397-B3-050/CF), and sequence is as follows:
Note: The underlined portion is the B7-H3 extracellular region; the italic portion is the His-tag marker.
Example 2. Screening positive sequence(s) for specific binding to human B7-H3
B cells were isolated from human PBMC, spleen, and lymph node tissues, and RNA was extracted to construct naive scFv phage antibody library (capacity 3.2x100). The constructed naive scFv phage antibody library was packaged to form phage particles, and then subjected to panning by liquid phase method. The phage was bound to the biotinylated B7-H3 in liquid phase, and then was separated by streptavidin magnetic beads. In order to obtain positive sequence(s) binding to human B7-H3 (R&D cat# 1949-B3-050/CF), biotinylated human B7-H3 was used for panning, and 500 monoclonal colonies were picked and packaged into phage scFv antibodies for phage ELISA testing. The binding activity of monoclonal phage to human B7-H3 (R&D cat# 1949-B3-050/CF) and murine B7-H3 (R&D cat#1397-B3-050/CF) were tested separately: 1 pg/ml human B7-H3 or murine B7-H3 and 1% BSA were coated on ELISA plate, phage supernatant diluted at 1:1 with blocking buffer was added, and detected with anti-M13 HRP; the clones with ELISA OD450 value of greater than 0.5, and with ratios of ELISA OD450 values for binding with human or murine B7-H3 to ELISA OD450 value for binding with 1% BSA greater than 2.0 were selected, and 9 clones were obtained.
Example 3. Construction of full length monoclonal antibodies
Full length antibodies were constructed for these 9 clones obtained by phage library screening, and then two antibodies (hl702 and h703, respectively) were confirmed to have strong affinity by ELISA binding assay. The process of constructing full length monoclonal antibody was as follows:
Based on the scFv antibody sequence(s) obtained by phase screening, primers were designed to construct the VH/VK/VL gene fragment of each single-chain antibody sequence by PCR. The heavy and light chain variable regions of hl702 and hl703 were obtained.
>hl702 heavy chain variable region sequence
QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVAVISYDGS NKYYVDSVKGRFTUSRDNSKNTLYLQMNSLRAEDTAVYYCARSARLYASFDYWGQG ALVTVSS SEQ ID NO: 6
>hl702 light chain variable region sequence
QTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRMLIYNTNTRS SGVPDRFSGSILGNKAALTITGAQADDESDYYCAIIHVDRDIWVFGGGTKLTVL SEQ ID NO: 7
>hl703 heavy chain variable region sequence
QVQLQESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGG STYYADSVKGRYTISRDNSKNTLYLQMNSLRAEDTAVYYCAKGVGPVHALDVWGQG TTVTVSS SEQ ID NO: 8
>hl703 light chain variable region sequence
DIRLTQSPSSLSASVGDRVTITCRASOSISTYLNWYQQKPGKAPILLINAVSGLQSGVP SRFSGSGSG THFTLTITSLQPEDFATYYCOQSYSTPMWT FGQGT KVEIK SEQ ID NO: 9
Note: The order is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, the italics in sequence are FR sequences, and the CDR sequences are underlined.
The CDR sequences in the light chain and heavy chain of each antibody are shown in Table 1.
Table 1. CDR regions of each heavy and light chain
Antibody HC LC
GFIFSSSA SGSVSTSHY HCDR1 SEQ ID NO: 10 LCDR1 SEQ ID NO: 13
ARSARLYASFDY AIHVDRDIWV HCDR3 LCDR3 SEQ ID NO: 12 SEQ ID NO: 15
GFTFSSYA QSISTY HCDR1 LCDR1 SEQ ID NO: 16 SEQ ID NO: 19 1703 ISGSGGST AVS HCDR2 LCDR2 SEQ ID NO: 17 SEQ ID NO: 20
AKGVGPVHALDV QQSYSTPMWT HCDR3 LCDR3 SEQ ID NO: 18 SEQ ID NO: 21
The antibody variable region was then homologously recombined with the constant region gene (CH1-FC/CL) fragment to construct the complete antibody VH-CH1-FC/VK-CL/VL-CL.
The constructed complete antibodies h1702-IgGl, h1703-IgG1 sequences are as follows:
h1702-IgGl:
h1702-IgG1 heavy chain amino acid sequence: (SEQ ID NO: 22)
h1702 light chain amino acid sequence: Lamada (SEQ ID NO: 23)
h1703-IgGl:
h1703-IgG1 heavy chain amino acid sequence: (SEQ ID NO: 24)
h1703 light chain amino acid sequence: Kappa (SEQ ID NO: 25)
In order to further improve the stability of the antibody, the amino acids of h702 light chain sequence were mutated, wherein the specific mutation involves that the first amino acid residue Q at N-terminus of the light chain was replaced by D, the first amino acid residue S at C-terminus was deleted, so as to obtain a more stable and uniform monoclonal antibody.
hl702-1 light chain amino acid sequence with mutation modification: (SEQ ID NO: 26)
Example 4. Expression and purification of fully human antibodies
The plasmids expressing the light and heavy chain of the antibody respectively were transfected into HEK293E cells at a ratio of 1.5:1, and the cell culture supernatant was collected 6 days later, and the cell debris was removed by high-speed centrifugation, and purification was performed by a Protein A column. The column was washed with PBS until the A280 reading dropped to the baseline. The target protein was eluted with an acidic eluent of pH 3.0 - pH 3.5 and neutralized with 1 M Tris-HCl, pH 8.0-9.0. The eluted sample was appropriately concentrated and further purified by gel chromatography Superdex 200 (GE) which was equilibrated by PBS to remove the aggregate. The monomer peak was collected and aliquoted for use.
The performance and beneficial effect of the antibodies of the present invention was tested by the following test methods:
Test example 1. ELISA binding assay
To test the binding ability of the screened B7-H3 antibodies to different forms of human B7-H3 in vitro, human 21g-B7-H3 (Cat.#1949-B3-050/CF, R&D) and human 41g-B7-H3 (Cat# 11188-H08H, Sino Biological) were used for binding assays in vitro.
Human B7-H3 protein (21g/4g) was diluted to a concentration of 1 pg/ml with PBS buffer, pH 7.4 (Sigma, P4417-100TAB), and added to a 96-well Elisa plate at a volume of 100 pl/well (Corning, CLS3590-100 EA), and placed at 4 °C overnight, for 16-20 hours. After discarding the liquid, 5% skim milk (GuangMing skim milk powder) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added at 120 pl/well, and the mixture was incubated at 37 °C for 2 hours to block. At the end of the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer. Then, 100pl/well of the corresponding B7-H3 antibodies with series dilution was added. The initial concentration was 1 M, then diluted with PBST buffer to 8 gradients, and incubated at 37 °C for 1 hour in the incubator. After incubation, the reaction solution in the plate was discarded, and the plate was washed 4 times with PBST. HRP-labeled goat anti-Human IgG Fcy fragment specific secondary antibody (Jackson Immuno Research, 109-005-008) diluted with PBST (1:4000) was added at 100pl/well, and incubated for 1 hour at 37 °C. After washing the plate 4 times with PBST, TMB chromogenic substrate (KPL, 52-00-03) was added at 100 pl/well, incubated for 3-5 min at room temperature, and 1 M H 2 SO4 was added at 100 pl/well to stop the reaction. The absorbance value was read using NOVOStar microplate reader at 450 nm, and the EC50 value for the binding between the antibody and the antigen was calculated. The results are shown in Table 2 , Figure 1 and Figure 2.
Table 2. The binding ability of different antibodies to human 2g-B7-H3 and 4g-B7-H3 antigen
EC50 human 21g-B7-H3(nM) human 41g-B7-H3(nM)
h1702 0.11 0.16
h1703 10.28 2.10
The results show that h1702 and h1703 have significant binding ability to both human 21g-B7-H3 and 4g-B7-H3, and h1702 has stronger binding ability.
Test example 2. Cross-binding assay to B7-H3 derived from different species
To test the binding ability of the screened B7-H3 antibodies to B7-H3 derived from different species in vitro, murine B7-H3 (Cat. #1397-B3-050/CF, R&D) was used for binding assays in vitro.
B7-H3 protein derived from different species (mouse B7-H3) was diluted to a concentration of 1 pg/ml with PBS buffer, pH 7.4 (Sigma, P4417-1OOTAB), added to a 96-well microtiter plate at a volume of 100 pl/well (Corning, CLS3590-100 EA), and placed at 4 °C overnight for 16-20 hours. After discarding the liquid, 5% skim milk (GuangMing skim milk powder) blocking solution diluted with PBST buffer (pH 7.4 PBS containing 0.05% Tween-20) was added at 120 pl/well, and the mixture was incubated at 37 °C for 2 hours to block. At the end of the blocking, the blocking solution was discarded, and the plate was washed 4 times with PBST buffer. Then, 100 pl/well of the corresponding B7-H3 antibodies at an initial concentration of 1 M were added, diluted with PBST buffer to 8 gradients, and incubated at 37 °C for 1 hour in the incubator. After incubation, the reaction solution in the plate was discarded, and the plate was washed 4 times with PBST, and HRP-labeled goat anti-Human IgG Fcy fragment specific secondary antibody (Jackson Immuno Research, 109-005-008) diluted with PBST (1:4000) was added at 100 pl/well, incubated for 1 hour at 37 °C. After washing the plate 4 times with PBST, TMB chromogenic substrate (KPL, 52-00-03) was added at 100 pl/well, incubated for 3-5 min at room temperature, and 1 M H 2 SO4 was added at 100 pl/well to stop the reaction, the absorbance value was read using NOVOStar microplate reader at 450 nm, the EC50 value for the binding between the antibody and the antigen was calculated (the results are shown in Table 3 and Figure 3).
Table 3. The binding ability of different antibodies to murine B7-H3 antigen
EC50 murine B7-H3 (nM)
h1702 18.12
h1703 86.68
The results shown that the binding ability of h1702 and h1703 to murine B7-H3 was weak, indicating that two monoclonal antibodies specifically bind to human B7-H3.
Test example 3. Biacore test for antibody affinity
The reaction affinity of anti-B7-H3 antibodies to human and murine B7-H3 was determined using a Biacore, GE instrument.
A biosensor chip Protein A (Cat. # 29127556, GE) was used to affinity capture a certain amount of antibody to be tested. A series diluted of human 2g-B7-H3 antigen (Cat. #1949- B3-050/CF, R&D), human 4g-B7-H3 antigen (Cat. #11188-HO8H, Sino Biological) or murine B7-H3 antigen (Cat. # 1397-B3-050/CF, R&D) was flowed through the surface of the chip. Real-time reaction signal was detected by using Biacore instrument (Biacore T200, GE), in order to obtain association and dissociation curves. After completion of each cycle of dissociation, the biochip was washed and regenerated with glycine-hydrochloric acid regeneration solution (pH 1.5) (Cat.#BR-1003-54, GE). The buffer used in the experiment was HBS-EP buffer solution (pH 7.4) (Cat.
# BR-1001-88, GE).
The experimental data was fitted with BIA evaluation version 4.1 GE software in a (1:1) Langmuir model to obtain affinity values. The experimental results are shown in Tables 4-6.
Table 4. The reaction affinity of different antibodies to human 2g-B7-H3 antigen
Antibody Antigen Affinity (M)
h1702 7.97E-7 human 21g-B7-H3 h1703 4.48E-7
Table 5. The reaction affinity of different antibodies to human 4g-B7-H3 antigen
Antibody Antigen Affinity (M)
h1702 8.55E-9 human 41g-B7-H3 hi703
Table 6. The reaction affinity of different antibodies to murine B7-H3 antigen
Antibody Antigen Affinity (M)
h1702 1.47E-6 murine B7-H3 h1703 5.02E-7
The affinity results of Biacore test show that h1702 has strong affinity to human
41g-B7-H3, reaching a level at nM, whereas the affinity to human 2g-B7-H3 or murine B7-H3 is relatively weaker; h1703 has weaker affinity to human 4g-B7-H3, human 21g-B7-H3 and murine B7-H3.
Test example 4. In vitro cell binding assay
In this experiment, the binding of antibody was evaluated based on the intensity of fluorescence signal of the antibody bond on the cell surface. After incubating 10 pg of primary antibody with 2x10 5 U87MG cells on ice for 30 minutes, excess antibody was removed by washing. The cells were incubated with APC anti-human IgG Fc (Biolegend, 409306) for 30 minutes at room temperature, and after removing excess antibody, the fluorescence signal on the cell surface was read using BD Verse (results were shown in Figure 4). The results indicate that h1702 specifically binds to tumor cell U87MG which overexpresses B7-H3.
Test example 5. In vitro endocytosis test
In this experiment, the endocytosis effect of the antibody was evaluated based on the intensity of fluorescence signal which was determined of the internalized antibody. The B7-H3 antibody and APC anti-human IgG Fc (Biolegend, 409306) were mixed at a molar ratio of 1:2 and incubated on ice for 15 minutes. After incubating the antibody mixture with 2x10 5 U87MG cells on ice for 30 minutes, excess antibody was removed by washing, and then the cells were transferred to a 37 °C prewarmed medium, and incubated at 37°C for 0, 15, 30, 60 and 120 minutes respectively. The cells were centrifuged and resuspended in the antibody elution buffer to get rid of antibodies. After incubating for 7 minutes at room temperature, the antibody elution buffer was removed by washing, and the intracellular fluorescence signal was read using BD Verse (results shown in Figure 5). The results show that both h1702 and h1703 were efficiently endocytosed into cells after binding to U87MG cell.
Test example 6. T1/2 evaluation on SD rats
4 SD rats (purchased from JSJ Experimental Animal Co., Ltd.), 2 males and 2 females, were maintained in light/dark cycle adjusted at 12/12 hours, with constant temperature of 24±3°C, humidity of 50-60%, and free access to food and water. On the day of the experiment, SD rats were injected with the test agent into tail vein at a dose of 3 mg/kg and an injection volume of 5 ml/kg.
The time for blood collection: On the first day of administration, blood was taken from ocular fundus vein at 5 min, 8 h, 24 h, 2 days, 3 days, 5 days, 8 days, and 15 days after administration, 200 pL each time (equivalent to 100 pL of serum); The collected blood samples were allowed to place at room temperature for half an hour until coagulation, and then centrifuged at 10,000 x g for 10 minutes at 4°C. The supernatant was collected and immediately stored at -80 °C. The B7-H3 antibody concentration in the serum was measured by ELISA, and PK analysis was performed. The results are shown in Table 7.
Table 7. T/2 of B7-H3 antibody in SD rats
Tested agent Route of administration T/2 (mean±SD, h)
h1702 IV (3 mg/kg ) 185±17
The results show that the half-life of the antibody of present invention in rats was approximately 185 h (7.7 days).
Test example 7. Physical stability of B7-H3 antibodies
DSC was used to detect the thermal stability of different antibodies, and the thermal stability in different buffer systems under different pH conditions was compared. Exemplary buffer systems corresponding to different pHs were 10 mM PB (pH 7) and 10 mM Acetate (pH 5.2). The sample was dissolved in the corresponding buffer, and the sample concentration was controlled at about 1 mg/ml. Detection was performed using MicroCal* VP-Capillary DSC (Malvern). Before detection, each sample and the blank buffer were degassed by a vacuum degasser for 1 to 2 minutes. 400 Pl of sample or blank buffer were added to each well of the sample plate (the load of instrument is 300 pl). The last two pairs of well-plates were respectively added with 14% Decon 90 and ddH 20 for cleaning. After the sample plate was loaded, a plastic soft cover was placed. The scanning temperature started from 25°C and ended at 100 °C. The scanning rate was 60 °C/h. The results are shown in Table 8. In several test systems, both h1702 and h1703 showed good thermal stability.
Table 8. DSC results of different antibodies
Sample Buffer Tm-onset (C) TM (C)
pH7.0 65.82 77.51 h1702 pH 5.2 65.59 78.97
pH7.0 61.33 73.19 h1703 pH 5.2 60.65 75.71
The purity of the sample was monitored by SEC-HPLC to investigate the stability under a certain concentration condition. As an example of condition, the sample concentration was controlled at about 40-50 mg/ml. The stability of different antibodies was compared in PBS (pH 7.4) system and pH 5.2 acetic acid/sucrose system at 4 °C, 30 °C, 40 °C for one month of storage. The purity of the antibody was examined using Xbridge protein BEH SEC 200A (Waters) HPLC column. After one month of investigation, both h1702 and h1703 showed good stability. The results are shown in Table 9.
Table 9. Stability results for different antibodies
Sample 4°C/month/purity 30°C/month/purity 40°C/month/purity
h1702/ acetic acid 99.25% 98.68% 97.85%
h1702/PBS 99.21% 98.07% 96.34%
h1703/ acetic acid 99.31% 99.04% 98.38%
h1703/PBS 99.18% 98.56% 96.99%
The results show that both h1702 and h1703 show excellent stability in both acetic acid and PBS buffers.
Test example 8. Chemical stability of B7-H3 antibodies
Chemical modification after antibody preparation is one of the common reasons leading to the product stability problem, especially the high degree of deamination, oxidation or isomerization modification at some amino acids in CDR region. Those modifications should be avoided or reduced. 500 pg of antibodies to be tested was dissolved in 500 Pl of PBS pH 7.4, and subjected to water bath at 40 °C; samples were taken at day 0, 10, and 20, respectively, for enzymatic hydrolysis experiments. 100 pg samples were taken at different time points and dissolved in 100 pl solution of 0.2 M His-HC, 8 M Gua-HCl, pH 6.0; 3 pl of 0.1 g/mL DTT was added, and subjected to water bath at 50 °C for 1 hour; Afterward, the samples were ultrafiltered twice with solution of 0.02 M His-HCl, pH 6.0, and 3 pL of 0.25 mg/mL trypsin was added. The mixture was hydrolyzed overnight at 37 °C in water bath. Potential modification sites were analyzed by mass spectrometry (the results are shown in Table 10) using Agilent 6530 Q-TOF The results show that h1702 and h1703 described in the present invention have no significantly increased trend towards deamidation, oxidation or heterogeneity, indicating that the molecules have excellent chemical stability.
Table 10. Chemical stability of different antibodies
Sample LC/HC position/modification DO D1O D20
LC M48/ oxidation 2.82% 2.9% 2.83%
h1702 M34/ oxidation 3.52% 3.46% 3.38% HC M83/ oxidation 0.98% 1.01% 0.01%
M34/ oxidation 1.93% 2.62% 2.16% h1703 HC M83/ oxidation 1.96% 2.62% 2.8%
Test example 9. Stability of h1702-1 antibody
The lambda type has one more amino acid S at the C-terminus than kappa type light chain, and the steric hindrance of the S may be a factor causing instability of interchain disulfide bond between light and heavy chain. The terminal amino acid S can be knocked out by molecular cloning, and the stability of the antibody under alkaline condition would be remarkably improved.
When the first amino acid at the N-terminus of the light chain of the naked antibody is Q, partial cyclization would also occur in the antibody, and which leads to an increase in charge heterogeneity of the sample, affecting on the stability of formulation and product. The first amino acid Q at N-terminus was mutated to D by molecular cloning, to eliminate the incomplete cyclization and significantly improve the antibody stability. The above modification did not significantly affect the affinity of the engineered antibody to its antigen.
The stability of h1702 and h1702-1 was tested by SEC, non-reducing CE-SDS analysis method (pH 9.0) and IEX analysis method.
SEC detection: Waters e2695 chromatograph and Xbridge BEH 200A SEC column were used. 50 pg antibody was loaded, and the elution was performed using PBS mobile phase in constant gradient.
CE-SDS NR method:
Samples were processed using the Beckman SDS-MW Analysis Kit. A buffer solution was added to 100 pg tested antibody was denatured by heating in sample buffer as described in manned protocol. Data was collected using PA800 capillary electrophoresis apparatus.
IEX method:
Waters Acquity H-Class chromatograph and Thermo MAbPac SCX-10 column were used. 50 pg of tested antibody was loaded, and a linear gradient was applied, using CX-1 pH Gradient Buffer Kit as the mobile phase; ultraviolet signal at a wavelength of 280 nm was collected.
Table 11. Comparison of stability of h1702 and h1702-1
SEC CE-SDS (pH9.0) IEX
h1702 100% 71.21% 40.5%
h1702-1 100% 94.67% 86.21%
Although the invention has been described in detail with the figures and specific embodiments for the purpose of a clear understanding, the description and embodiments should not be interpreted as limiting the scope of the present invention. All public contents of literatures and patents cited herein are expressly incorporated by reference in their entirety.
Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.
The reference in this specification to any prior publication (or information derived from it), or to any matter which is known, is not, and should not be taken as an acknowledgment or admission or any form of suggestion that that prior publication (or information derived from it) or known matter forms part of the common general knowledge in the field of endeavour to which this specification relates.
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Sequence listing Sequence listing
<110> JIANGSU HENGRUI MEDICINE CO.,LTD; SHANGHAI HENGRUI PHARMACEUTICAL <110> JIANGSU HENGRUI MEDICINE CO. LTD; SHANGHAI HENGRUI PHARMACEUTICAL CO.,LTD CO. LTD
<120> B7‐H3 ANTIBODY, ANTIGEN‐BINDING FRAGMENT THEREOF AND MEDICAL USE THEREOF <120> B7-H3 ANTIBODY, ANTIGEN-BINDING FRAGMENT THEREOF AND MEDICAL USE THEREOF
<130> 780019CPCT <130> 780019CPCT
<160> 26 <160> 26
<170> SIPOSequenceListing 1.0 <170> SIPOSequenceListing 1.0
<210> 1 <210> 1 <211> 534 <211> 534 <212> PRT <212> PRT <213> Homo sapiens <213> Homo sapiens
<220> <220> <221> PEPTIDE <221> PEPTIDE <223> Human B7H3 full‐length amino acid sequence <223> Human B7H3 full-length amino acid sequence
<400> 1 <400> 1 Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala Met Leu Arg Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala 1 5 10 15 1 5 10 15 Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln Ala Leu Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30 20 25 30 Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45 35 40 45 Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala 65 70 75 80 70 75 80 Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe 85 90 95 85 90 95 Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 100 105 110 Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp 115 120 125 115 120 125 Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr 145 150 155 160 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 165 170 175 Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 180 185 190 Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu 195 200 205 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 210 215 220 Page 1 Page 1
780019CPCTseqlisting.txt 780019CPCTseqlisting.1 txt Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Ile Thr Pro Gln 225 230 235 240 225 230 235 240 Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Glu Asp Pro Val 245 250 255 245 250 255 Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270 260 265 270 Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285 275 280 285 Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300 290 295 300 Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln 305 310 315 320 305 310 315 320 Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu Gly 325 330 335 325 330 335 Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser Ala Ala Val 340 345 350 340 345 350 Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu 355 360 365 355 360 365 Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Ile Thr Cys Ser 370 375 380 370 375 380 Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Gln Asp Gly Gln 385 390 395 400 385 390 395 400 Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415 405 410 415 Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425 430 420 425 430 Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp 435 440 445 435 440 445 Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro 450 455 460 450 455 460 Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu Glu Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu 465 470 475 480 465 470 475 480 Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys Leu Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys 485 490 495 485 490 495 Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly 500 505 510 500 505 510 Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys Glu Asp 515 520 525 515 520 525 Asp Gly Gln Glu Ile Ala Asp Gly Gln Glu Ile Ala 530 530
<210> 2 <210> 2 <211> 316 <211> 316 <212> PRT <212> PRT <213> Mus musculus <213> Mus musculus
<220> <220> <221> PEPTIDE <221> PEPTIDE <223> Murine B7H3 full‐length amino acid sequence <223> Murine B7H3 full-length amino acid sequence
<400> 2 <400> 2 Page 2 Page 2
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr Met Leu Arg Gly Trp Gly Gly Pro Ser Val Gly Val Cys Val Arg Thr 1 5 10 15 1 5 10 15 Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln Ala Leu Gly Val Leu Cys Leu Cys Leu Thr Gly Ala Val Glu Val Gln 20 25 30 20 25 30 Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Asp Ala Thr Leu 35 40 45 35 40 45 Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60 50 55 60 Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr 65 70 75 80 70 75 80 Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg Thr Ala Leu Phe Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg Thr Ala Leu Phe 85 90 95 85 90 95 Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val 100 105 110 100 105 110 Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln Asp Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Ser Ile Gln Asp 115 120 125 115 120 125 Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140 130 135 140 Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn Met Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asn Met 145 150 155 160 145 150 155 160 Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175 165 170 175 Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185 190 180 185 190 Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His Ser Val Leu Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val His Ser Val Leu 195 200 205 195 200 205 Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn 210 215 220 210 215 220 Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln 225 230 235 240 225 230 235 240 Pro Leu Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser Pro Leu Thr Phe Pro Pro Glu Ala Leu Trp Val Thr Val Gly Leu Ser 245 250 255 245 250 255 Val Cys Leu Val Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg Val Cys Leu Val Val Leu Leu Val Ala Leu Ala Phe Val Cys Trp Arg 260 265 270 260 265 270 Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Lys Ile Lys Gln Ser Cys Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln 275 280 285 275 280 285 Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys Pro Asp Gly Asp Gly Glu Gly Ser Lys Thr Ala Leu Arg Pro Leu Lys Pro 290 295 300 290 295 300 Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala Ser Glu Asn Lys Glu Asp Asp Gly Gln Glu Ile Ala 305 310 315 305 310 315
<210> 3 <210> 3 <211> 223 <211> 223 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> PEPTIDE <221> PEPTIDE <223> Human B7H3 antigen: 2Ig‐B7H3 <223> Human B7H3 antigen: 2Ig-B7H3
<400> 3 <400> 3 Page 3 Page 3
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr 1 5 10 15 1 5 10 15 Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu 20 25 30 20 25 30 Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val 35 40 45 35 40 45 His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg 50 55 60 50 55 60 Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg 65 70 75 80 70 75 80 Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val 85 90 95 85 90 95 Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala 100 105 110 100 105 110 Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg 115 120 125 115 120 125 Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro 130 135 140 130 135 140 Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly 145 150 155 160 145 150 155 160 Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val 165 170 175 165 170 175 His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys 180 185 190 180 185 190 Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr 195 200 205 195 200 205 Ile Thr Pro Gln Arg Ser Pro Thr Gly His His His His His His Ile Thr Pro Gln Arg Ser Pro Thr Gly His His His His His His 210 215 220 210 215 220
<210> 4 <210> 4 <211> 439 <211> 439 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> PEPTIDE <221> PEPTIDE <223> Human B7H3 antigen: 4Ig‐B7H3 <223> Human B7H3 antigen: 4Ig-B7H3
<400> 4 <400> 4 Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Leu Glu Val Gln Val Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr 1 5 10 15 1 5 10 15 Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Asp Ala Thr Leu Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu 20 25 30 20 25 30 Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val 35 40 45 35 40 45 His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg His Ser Phe Ala Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg 50 55 60 50 55 60 Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg 65 70 75 80 70 75 80 Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Leu Gln Arg Val Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val 85 90 95 85 90 95 Page 4 Page 4
780019CPCTseqlisting.txt 780019CPCTseqlisting. txt Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala Ser Ile Arg Asp Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala Ala 100 105 110 100 105 110 Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg 115 120 125 115 120 125 Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Pro Gly Asp Thr Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro 130 135 140 130 135 140 Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Glu Ala Glu Val Phe Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly 145 150 155 160 145 150 155 160 Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val Asn Val Thr Thr Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val 165 170 175 165 170 175 His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys His Ser Ile Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys 180 185 190 180 185 190 Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr 195 200 205 195 200 205 Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro Ile Thr Pro Gln Arg Ser Pro Thr Gly Ala Val Glu Val Gln Val Pro 210 215 220 210 215 220 Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu Arg Cys 225 230 235 240 225 230 235 240 Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn Leu Ile 245 250 255 245 250 255 Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Thr Glu Gly 260 265 270 260 265 270 Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Arg Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp 275 280 285 275 280 285 Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val 290 295 300 290 295 300 Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly 305 310 315 320 305 310 315 320 Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser Ser Ala Ala Val Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro Ser 325 330 335 325 330 335 Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr 340 345 350 340 345 350 Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp Ile Thr Cys Ser Ser Tyr Arg Gly Tyr Pro Glu Ala Glu Val Phe Trp 355 360 365 355 360 365 Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr Ser Gln 370 375 380 370 375 380 Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Val Leu Arg Val 385 390 395 400 385 390 395 400 Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val 405 410 415 405 410 415 Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Leu Gln Gln Asp Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met 420 425 430 420 425 430 Thr His His His His His His Thr His His His His His His 435 435
<210> 5 <210> 5 <211> 222 <211> 222 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> PEPTIDE <221> PEPTIDE Page 5 Page 5
780019CPCTseqlisting.txt 780019CPCTseqlisting. txt <223> Murine B7H3 antigen <223> Murine B7H3 antigen
<400> 5 <400> 5 Val Glu Val Gln Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr Val Glu Val Gln Val Ser Glu Asp Pro Val Val Ala Leu Val Asp Thr 1 5 10 15 1 5 10 15 Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Asp Ala Thr Leu Arg Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu 20 25 30 20 25 30 Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val 35 40 45 35 40 45 His Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg His Ser Phe Thr Glu Gly Arg Asp Gln Gly Ser Ala Tyr Ser Asn Arg 50 55 60 50 55 60 Thr Ala Leu Phe Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg Thr Ala Leu Phe Pro Asp Leu Leu Val Gln Gly Asn Ala Ser Leu Arg 65 70 75 80 70 75 80 Leu Gln Arg Val Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val Leu Gln Arg Val Arg Val Thr Asp Glu Gly Ser Tyr Thr Cys Phe Val 85 90 95 85 90 95 Ser Ile Gln Asp Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala Ser Ile Gln Asp Phe Asp Ser Ala Ala Val Ser Leu Gln Val Ala Ala 100 105 110 100 105 110 Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg Pro Tyr Ser Lys Pro Ser Met Thr Leu Glu Pro Asn Lys Asp Leu Arg 115 120 125 115 120 125 Pro Gly Asn Met Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Pro Gly Asn Met Val Thr Ile Thr Cys Ser Ser Tyr Gln Gly Tyr Pro 130 135 140 130 135 140 Glu Ala Glu Val Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly Glu Ala Glu Val Phe Trp Lys Asp Gly Gln Gly Val Pro Leu Thr Gly 145 150 155 160 145 150 155 160 Asn Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val Asn Val Thr Thr Ser Gln Met Ala Asn Glu Arg Gly Leu Phe Asp Val 165 170 175 165 170 175 His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys His Ser Val Leu Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys 180 185 190 180 185 190 Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala His Gly Ser Val Thr 195 200 205 195 200 205 Ile Thr Gly Gln Pro Leu Thr Phe His His His His His His Ile Thr Gly Gln Pro Leu Thr Phe His His His His His His 210 215 220 210 215 220
<210> 6 <210> 6 <211> 119 <211> 119 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 Heavy chain variable region <223> h1702 Heavy chain variable region
<400> 6 <400> 6 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 50 55 60 Page 6 Page 6
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 100 105 110 Ala Leu Val Thr Val Ser Ser Ala Leu Val Thr Val Ser Ser 115 115
<210> 7 <210> 7 <211> 110 <211> 110 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 light chain variable region <223> h1702 light chain variable region
<400> 7 <400> 7 Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 100 105 110
<210> 8 <210> 8 <211> 119 <211> 119 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 Heavy chain variable region <223> h1703 Heavy chain variable region
<400> 8 <400> 8 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 35 40 45 Page 7 Page 7
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 50 55 60 Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95 Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly 100 105 110 100 105 110 Thr Thr Val Thr Val Ser Ser Thr Thr Val Thr Val Ser Ser 115 115
<210> 9 <210> 9 <211> 108 <211> 108 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 light chain variable region <223> h1703 light chain variable region
<400> 9 <400> 9 Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile 35 40 45 35 40 45 Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro 65 70 75 80 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met 85 90 95 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 100 105
<210> 10 <210> 10 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 HCDR1 <223> h1702 HCDR1
<400> 10 <400> 10 Gly Phe Ile Phe Ser Ser Ser Ala Gly Phe Ile Phe Ser Ser Ser Ala 1 5 1 5
<210> 11 <210> 11 Page 8 Page 8
780019CPCTseqlisting.txt 780019CPCTseqlisting.tx <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 HCDR2 <223> h1702 HCDR2
<400> 11 <400> 11 Ile Ser Tyr Asp Gly Ser Asn Lys Ile Ser Tyr Asp Gly Ser Asn Lys 1 5 1 5
<210> 12 <210> 12 <211> 12 <211> 12 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 HCDR3 <223> h1702 HCDR3
<400> 12 <400> 12 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr 1 5 10 1 5 10
<210> 13 <210> 13 <211> 9 <211> 9 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 LCDR1 <223> h1702 LCDR1
<400> 13 <400> 13 Ser Gly Ser Val Ser Thr Ser His Tyr Ser Gly Ser Val Ser Thr Ser His Tyr 1 5 1 5
<210> 14 <210> 14 <211> 3 <211> 3 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 LCDR2 <223> h1702 LCDR2
<400> 14 <400> 14 Page 9 Page 9
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt Asn Thr Asn Asn Thr Asn 1 1
<210> 15 <210> 15 <211> 10 <211> 10 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <223> h1702 LCDR3 <223> h1702 LCDR3
<400> 15 <400> 15 Ala Ile His Val Asp Arg Asp Ile Trp Val Ala Ile His Val Asp Arg Asp Ile Trp Val 1 5 10 1 5 10
<210> 16 <210> 16 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 HCDR1 <223> h1703 HCDR1
<400> 16 <400> 16 Gly Phe Thr Phe Ser Ser Tyr Ala Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 1 5
<210> 17 <210> 17 <211> 8 <211> 8 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 HCDR2 <223> h1703 HCDR2
<400> 17 <400> 17 Ile Ser Gly Ser Gly Gly Ser Thr Ile Ser Gly Ser Gly Gly Ser Thr 1 5 1 5
<210> 18 <210> 18 <211> 12 <211> 12 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 HCDR3 <223> h1703 HCDR3 Page 10 Page 10
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt
<400> 18 <400> 18 Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val 1 5 10 1 5 10
<210> 19 <210> 19 <211> 6 <211> 6 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 LCDR1 <223> h1703 LCDR1
<400> 19 <400> 19 Gln Ser Ile Ser Thr Tyr Gln Ser Ile Ser Thr Tyr 1 5 1 5
<210> 20 <210> 20 <211> 3 <211> 3 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 LCDR2 <223> h1703 LCDR2
<400> 20 <400> 20 Ala Val Ser Ala Val Ser 1 1
<210> 21 <210> 21 <211> 10 <211> 10 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 LCDR3 <223> h1703 LCDR3
<400> 21 <400> 21 Gln Gln Ser Tyr Ser Thr Pro Met Trp Thr Gln Gln Ser Tyr Ser Thr Pro Met Trp Thr 1 5 10 1 5 10
<210> 22 <210> 22 <211> 449 <211> 449 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence Page 11 Page 11
780019CPCTseqlisting.txt 780019CPCTseqlisting.t
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702‐IgG1 heavy chain <223> h1702-IgG1 heavy chain
<400> 22 <400> 22 Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser 20 25 30 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 35 40 45 Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val 50 55 60 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95 Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly 100 105 110 100 105 110 Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Page 12 Page 12
780019CPCTseqlisting.txt 780019CPCTseqlisting. txt 355 360 365 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 435 440 445 Lys Lys
<210> 23 <210> 23 <211> 216 <211> 216 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702 light chain <223> h1702 light chain
<400> 23 <400> 23 Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly Gln Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 195 200 205 Thr Val Ala Pro Thr Glu Cys Ser Thr Val Ala Pro Thr Glu Cys Ser Page 13 Page 13
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt 210 215 210 215
<210> 24 <210> 24 <211> 449 <211> 449 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703‐IgG1 heavy chain <223> h1703-IgG1 heavy chain
<400> 24 <400> 24 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 35 40 45 Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val Ser Ala Ile Ser Gly Ser Gly Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 50 55 60 Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Lys Gly Arg Tyr Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 85 90 95 Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly Ala Lys Gly Val Gly Pro Val His Ala Leu Asp Val Trp Gly Gln Gly 100 105 110 100 105 110 Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val 290 295 300 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Page 14 Page 14
780019CPCTseqlisting.txt 780019CPCTseqlisting.1 txt 305 310 315 320 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 340 345 350 Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 435 440 445 Lys Lys
<210> 25 <210> 25 <211> 215 <211> 215 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1703 light chain <223> h1703 light chain
<400> 25 <400> 25 Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly Asp Ile Arg Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Thr Tyr 20 25 30 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Ile Leu Leu Ile 35 40 45 35 40 45 Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly Asn Ala Val Ser Gly Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 50 55 60 Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro Ser Gly Ser Gly Thr His Phe Thr Leu Thr Ile Thr Ser Leu Gln Pro 65 70 75 80 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro Met 85 90 95 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala 100 105 110 100 105 110 Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser 115 120 125 115 120 125 Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu 130 135 140 130 135 140 Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser 145 150 155 160 145 150 155 160 Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Page 15 Page 15
780019CPCTseqlisting.txt 780019CPCTseqlisting.txt 165 170 175 165 170 175 Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val 180 185 190 180 185 190 Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys 195 200 205 195 200 205 Ser Phe Asn Arg Gly Glu Cys Ser Phe Asn Arg Gly Glu Cys 210 215 210 215
<210> 26 <210> 26 <211> 215 <211> 215 <212> PRT <212> PRT <213> Artificial Sequence <213> Artificial Sequence
<220> <220> <221> DOMAIN <221> DOMAIN <223> h1702‐1 light chain <223> h1702-1 light chain
<400> 26 <400> 26 Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly 1 5 10 15 1 5 10 15 Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser 20 25 30 20 25 30 His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met 35 40 45 35 40 45 Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe 50 55 60 50 55 60 Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala 65 70 75 80 70 75 80 Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg 85 90 95 85 90 95 Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln 100 105 110 100 105 110 Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu 115 120 125 115 120 125 Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr 130 135 140 130 135 140 Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys 145 150 155 160 145 150 155 160 Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr 165 170 175 165 170 175 Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His 180 185 190 180 185 190 Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys 195 200 205 195 200 205 Thr Val Ala Pro Thr Glu Cys Thr Val Ala Pro Thr Glu Cys 210 215 210 215
Page 16 Page 16
Claims (20)
1. A B7-H3 antibody or antigen-binding fragment thereof, which binds to human B7-H3, wherein the B7-H3 antibody or antigen-binding fragment thereof comprises: an antibody heavy chain variable region comprising an HCDR1, HCDR2 and HCDR3 comprising sequences as shown in SEQ ID NO: 10, 11 and 12, respectively; and an antibody light chain variable region comprising an LCDR1, LCDR2 and LCDR3 comprising sequences as shown in SEQ ID NO: 13, 14 and 15, respectively.
2. The B7-H3 antibody or antigen-binding fragment thereof according to claim 1, wherein the monoclonal antibody is a recombinant antibody.
3. The B7-H3 antibody or antigen-binding fragment thereof according to claim 2, wherein the monoclonal antibody is a human recombinant antibody or antigen-binding fragment thereof.
4. The B7-H3 antibody or antigen-binding fragment thereof according to claim 3, wherein the framework (FR) sequences of light chain and heavy chain variable regions of the human recombinant antibody are derived from a human germline light chain and heavy chain, respectively, or mutant sequence thereof.
5. The B7-H3 antibody or antigen-binding fragment thereof according to claim 4, wherein the human recombinant antibody comprises a heavy chain variable region as shown in SEQ ID NO: 6 or variant thereof; wherein the variant has deletion, substitution or addition of 1-10 amino acids in the heavy chain variable region of SEQ ID NO: 6.
6. The B7-H3 antibody or antigen-binding fragment thereof according to claim 4, wherein the human recombinant antibody comprises a light chain variable region as shown in SEQ ID NO: 7 or variant thereof; wherein the variant has deletion, substitution or addition of 1-10 amino acids in the light chain variable region of SEQ ID NO: 7.
7. The B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the B7-H3 antibody further comprises a human antibody constant region, preferably the B7-H3 antibody is a full-length antibody consisting of the heavy chain and light chain as shown in SEQ ID NO: 22 and 23, respectively, or a full-length antibody consisting of the heavy chain and light chain as shown in SEQ ID NO: 22 and 26, respectively.
8. The B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, wherein the antigen-binding fragment is selected from the group consisting of Fab, Fab', F(ab')2, single-chain antibody (scFv), dimerized V region (diabody), disulfide-stabilized V region (dsFv) and CDR-containing peptide.
9. A pharmaceutical composition comprising a therapeutically effective amount of the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, and one or more pharmaceutically acceptable carriers, diluents or excipients.
10. A nucleic acid molecule, encoding the antibody or antigen-binding fragment thereof according to any one of claims I to 8.
11. A recombinant vector, comprising the nucleic acid molecule of claim 10.
12. A host cell transformed with the recombinant vector according to claim 11, wherein the host cell is selected from the group consisting of prokaryotic cell and eukaryotic cell, preferably eukaryotic cell, more preferably mammalian cell or yeast cell.
13. A method for producing the antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, wherein the method comprises: cultivating the host cell of claim 12 in a culture to form and accumulate the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims I to 8, and recovering the accumulated antibody or antigen-binding fragment thereof from the culture.
14. A method for immunologically detecting or measurement of B7-H3, wherein the method comprises a step of detecting B7-H3 by using the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.
15. A reagent for detecting or measurement of human B7-H3, wherein the reagent comprises the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.
16. A diagnostic reagent for detecting diseases related to human B7-H3 positive cells, wherein the diagnostic reagent comprises the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims I to 8.
17. A method for diagnosing diseases related to human B7-H3 positive cells, the method comprises a step of detecting or determining B7-H3 or B7-H3 positive cells by using the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8.
18. A therapeutic agent for treating diseases related to B7-H3 positive cells, the therapeutic agent comprises the B7-H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 8, or the pharmaceutical composition according to claim 9 or the nucleic acid molecule of claim 10.
19. A method for treating diseases related to B7-H3 positive cells, wherein the method comprises inducing death of B7-H3 positive cells by utilizing the B7-H3 antibody or antigen-binding fragment thereof of any one of claims 1 to 8, or the pharmaceutical composition of claim 9.
20. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 8 in the preparation of a therapeutic agent for treating diseases related to human B7-H3 positive cells.
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| AU2018243123B2 (en) | 2017-03-31 | 2025-03-13 | Changzhou Hansoh Pharmaceutical Co., Ltd. | B7-H3 antibody, antigen-binding fragment thereof and medical use thereof |
| CN109963591B (en) * | 2017-08-04 | 2023-04-04 | 江苏恒瑞医药股份有限公司 | B7H3 antibody-drug conjugate and medical application thereof |
| US10916053B1 (en) | 2019-11-26 | 2021-02-09 | Sdc U.S. Smilepay Spv | Systems and methods for constructing a three-dimensional model from two-dimensional images |
| US11270523B2 (en) | 2017-11-29 | 2022-03-08 | Sdc U.S. Smilepay Spv | Systems and methods for constructing a three-dimensional model from two-dimensional images |
| US11403813B2 (en) | 2019-11-26 | 2022-08-02 | Sdc U.S. Smilepay Spv | Systems and methods for constructing a three-dimensional model from two-dimensional images |
| WO2020018964A1 (en) | 2018-07-20 | 2020-01-23 | Fred Hutchinson Cancer Research Center | Compositions and methods for controlled expression of antigen-specific receptors |
| UA129564C2 (en) * | 2018-09-30 | 2025-06-04 | Джянгсу Хансо Фармасьютікал Груп Ко., Лтд. | ANTI-B7H3 ANTIBODY-EXATECAN ANALOGUE CONJUGATE AND ITS MEDICAL APPLICATION |
| EP3880712A4 (en) * | 2018-11-16 | 2022-11-16 | Albert Einstein College of Medicine | MONOCLONAL ANTIBODIES TO THE IGV DOMAIN OF B7-H3 AND USES THEREOF |
| WO2020114479A1 (en) * | 2018-12-07 | 2020-06-11 | 江苏恒瑞医药股份有限公司 | Multispecific protein molecule |
| US11030801B2 (en) | 2019-05-17 | 2021-06-08 | Standard Cyborg, Inc. | Three-dimensional modeling toolkit |
| US12056820B2 (en) | 2019-05-17 | 2024-08-06 | Sdc U.S. Smilepay Spv | Three-dimensional modeling toolkit |
| KR102732027B1 (en) * | 2019-07-09 | 2024-11-20 | 주식회사 와이바이오로직스 | Antibody specifically binding to B7-H3(CD276) and Use thereof |
| WO2021052307A1 (en) * | 2019-09-16 | 2021-03-25 | 南京圣和药业股份有限公司 | Anti-b7-h3 antibody and application thereof |
| AU2021297036A1 (en) | 2020-06-26 | 2023-01-19 | Intocell, Inc. | Antibody-drug conjugates comprising anti-b7-h3 antibodies |
| CN120093941A (en) | 2021-02-09 | 2025-06-06 | 苏州宜联生物医药有限公司 | Bioactive substance conjugate, preparation method and application thereof |
| US20240158505A1 (en) * | 2021-03-26 | 2024-05-16 | Theranotics Co., Ltd. | B7-h3 antibody or antigen-binding fragment thereof, and use thereof |
| WO2023155808A1 (en) | 2022-02-16 | 2023-08-24 | 苏州宜联生物医药有限公司 | Conjugate of antibody-eribulin or derivative thereof, intermediate thereof, preparation method therefor, pharmaceutical composition thereof and use thereof |
| CN116903743B (en) * | 2022-02-25 | 2024-01-05 | 南京蓬勃生物科技有限公司 | Antibodies against human B7-H3 and variants thereof |
| CN119212733A (en) | 2022-05-18 | 2024-12-27 | 苏州宜联生物医药有限公司 | Antibody drug conjugate containing protein degrader-type bioactive compounds and preparation method and use thereof |
| JP2025517737A (en) * | 2022-05-18 | 2025-06-10 | ウーシー バイオロジクス アイルランド リミテッド | Anti-B7H3 antibodies and uses thereof |
| WO2024106939A1 (en) * | 2022-11-15 | 2024-05-23 | 주식회사 셀랩메드 | Antibody specifically binding to b7-h3 |
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| WO2011109400A2 (en) * | 2010-03-04 | 2011-09-09 | Macrogenics,Inc. | Antibodies reactive with b7-h3, immunologically active fragments thereof and uses thereof |
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| WO2011109400A2 (en) * | 2010-03-04 | 2011-09-09 | Macrogenics,Inc. | Antibodies reactive with b7-h3, immunologically active fragments thereof and uses thereof |
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| US11680100B2 (en) | 2023-06-20 |
| AU2018243123A1 (en) | 2019-09-26 |
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| JP7158403B2 (en) | 2022-10-21 |
| RU2019132843A (en) | 2021-04-30 |
| CN109937212A (en) | 2019-06-25 |
| CN109937212B (en) | 2022-06-21 |
| CA3056474A1 (en) | 2018-10-04 |
| US20200031934A1 (en) | 2020-01-30 |
| TW201837056A (en) | 2018-10-16 |
| MX2019011117A (en) | 2019-11-05 |
| JP2020515251A (en) | 2020-05-28 |
| EP3604337A1 (en) | 2020-02-05 |
| US10899837B2 (en) | 2021-01-26 |
| RU2019132843A3 (en) | 2021-08-16 |
| KR20240063177A (en) | 2024-05-09 |
| TWI796328B (en) | 2023-03-21 |
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